T-DNA mediated Gene Transfer in Plants (T-DNA 는 식물에서 유전자 이동을 중재했다 )

Sathiyaraj Srinivasan

사티야라지 스리니바산

Dept. of Oriental Medicinal materials and Processing

Kyung Hee University,

Republic of Korea – 449 701

Gene Transfer Technique

To transfer a gene from one DNA molecule to another DNA molecule.

Gene Transfer includes isolation of gene, manipulation (target gene insertion ) and reintroduction of DNA into the cells or model organisms.

It used to make a crop to resistant to a particular herbicide, pest, weeds or introducing a novel trait, or producing a new protein or enzyme.

The gene transfer methods normally include three categories:

1. transduction by biochemical methods, 2. transduction by physical methods, 3. virus-mediated transduction.

The gene transfer results can be transient and stable transduction.

Transfection Types:

Transient transfection

In transient transfection, the transfected DNA is not integrated into host chromosome. DNA is transferred into a recipient cell in order to obtain a temporary but high level of expression of the target gene.

Fast, flexible, not influenced by position effects, can provide large amounts of protein for detailed characterization, does not require regeneration system, gene expression limited to specific plant tissues.

Stable transfection

Stable transfection is also called permanent transfection. By the stable transfection, the transferred DNA is integrated (inserted) into chromosomal DNA and the genetics of recipient cells is permanent changed.

Time consuming, labor intensive, influenced by position effects, large production volumes, requires regeneration system, large amounts of glasshouse space, gene expression in all plant tissues

Transformation Methods:

Generally, there are 9 ways for gene transfer: (as published by Sambrook, 2001).

(1) Lipid-mediated method,

(2) Calcium-phosphate mediated,

(3) DEAE-dextran-mediated,

(4) Electroporation,

(5) Biolistics,

(6) Viral vectors,

(7) Polybrene,

(8) Laser transfection,

(9) Gene transfection enhanced by elevated temperature.

Introduction to Agrobacterium tumefaciens

Agrobacterium tumefaciensAgrobacterium tumefaciens is a ubiquitous soil borne pathogen is a ubiquitous soil borne pathogen responsible for Crown Gall disease, affecting many higher species of plant.responsible for Crown Gall disease, affecting many higher species of plant.

It’s a GIt’s a Gram negative, motile, rod shaped bacterium which is non ram negative, motile, rod shaped bacterium which is non sporing, and is closely related to the N-fixing rhizobium bacteria which form root sporing, and is closely related to the N-fixing rhizobium bacteria which form root nodules on leguminous plants.nodules on leguminous plants.

The bacterium is surrounded by a small number of peritricious flagella.

Virulent bacteria contain one or more large plasmids, one of which Virulent bacteria contain one or more large plasmids, one of which carries the genes for tumour induction and is known as the Ti (tumour inducing) carries the genes for tumour induction and is known as the Ti (tumour inducing) plasmid.plasmid.

Agrobacterium tumefaciens

Crown Gall Disease

Mechanism of infection of Bacterium in plants

1. Nopaline plasmids: carry gene for synthesizing nopaline in the plant and for utilization (catabolism) in the bacteria. Tumors can differentiate into shooty masses (teratomas).

2. Octopine plasmids: carry genes (3 required) to synthesize octopine in the plant and catabolism in the bacteria. Tumors do not differentiate,

but remain as callus tissue.3. Agropine plasmids: carry genes for agropine synthesis and catabolism. Tumors do not differentiate and die out.

4. Ri plasmids: induce hairy root disease on some plants and crown gall on others; have agropine-type genes and may have segments from both nopaline and octopine plasmids

Ti plasmids can be classified according to the opines producedTi plasmids can be classified according to the opines produced

Ti PlasmidTi Plasmid ::

The The tumor inducingtumor inducing plasmid ( plasmid (pTipTi) contains a portion that is ) contains a portion that is transferred to the plant cell is Transfer DNA (transferred to the plant cell is Transfer DNA (T-DNAT-DNA))

Vir Genes and their Functions

Vir GeneVir Gene FunctionFunctionVir A, Vir A,

Vir GVir G

Sense phenolic compounds from wounded plant cells and induce Sense phenolic compounds from wounded plant cells and induce expression of other virulence genes expression of other virulence genes

VirD2 VirD2 Endonuclease; cuts T-DNA at right border to initiate T-strand synthesis Endonuclease; cuts T-DNA at right border to initiate T-strand synthesis

Vir D1 Vir D1 Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp border sequence border sequence

Vir D2 Vir D2 Covalently attaches to the 5I end of the T-strand, thus forming the T-DNA Covalently attaches to the 5I end of the T-strand, thus forming the T-DNA Complex. Also guides the T-DNA complex through the nuclear pores Complex. Also guides the T-DNA complex through the nuclear pores

Vir C Vir C Binds to the 'overdrive' region to promote high efficiency T-strand Binds to the 'overdrive' region to promote high efficiency T-strand Synthesis Synthesis

Vir E2 Vir E2 Binds to T-strand protecting it from nuclease attack, and intercalates with Binds to T-strand protecting it from nuclease attack, and intercalates with lipids to form channels in the plant membranes through which thelipids to form channels in the plant membranes through which theT-complex passes T-complex passes

Vir E1 Vir E1 Acts as a chaperone which stabilises Vir E2 in the Acts as a chaperone which stabilises Vir E2 in the AgrobacteriumAgrobacterium

Vir B & Vir Vir B & Vir D4 D4

Assemble into a secretion system which spans the inner and outer Assemble into a secretion system which spans the inner and outer bacterial membranes. Required for Export of the T-complex and Vir E2 bacterial membranes. Required for Export of the T-complex and Vir E2 into the plant cell into the plant cell

1. Cytokinins (plant hormone for cell plant division and tumorous growth)

2. Enzymes for indoleacetic acid (auxin) synthesisAnother plant hormone (inducing stem and leaf elongation, inducing parthenocarpy and

preventing aging)

3. Enzymes for synthesis and release of novel plant metabolites:

the opines (uniques amino acid derivatives) the agrocinopines (phosphorylated sugar derivatives) .

Opines and agrocinopines are NUTRIENTS for A.tumefacies. They can not be used by other bacterial species

It provides unique niche for A.tumefaciens

Important genes encoded by Important genes encoded by Ti plasmidTi plasmid

Nopaline

1. Agrobacterium tumefaciens chromosomal genes: chvA, chvB, pscA required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface.

2. Virulence region (vir) carried on pTi, but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer.

3. T-DNA encodes genes for opine synthesis and for tumor production.

4. oc (opine catabolism) genes carried on the pTi and allows the

bacterium to utilize opines as nutrient.

Ti plasmids and the bacterial chromosome act in concert Ti plasmids and the bacterial chromosome act in concert to transform the plantto transform the plant

T-DNA

vir genesopine catabolism

pTipTitra

transfer to the plant

bacterial conjugation

Agrobacterium chromosomal DNAAgrobacterium chromosomal DNA

chvAchvB

pscA

oriV inc pTi’s are in the same inc group.

Ti PlasmidTi Plasmid

Tumor-producing genes

Virulence region

Opine catabolism

ORI

T-DNAregion

DNA between L and R borders istransferred to plantas ssDNA;

T-DNA encoded genes can be substituted by target genes

The basis of The basis of AgrobacteriumAgrobacterium-mediated genetic -mediated genetic engineeringengineering

T-DNA of T-DNA of A. tumefaciensA. tumefaciens is excised and integrates into is excised and integrates into the plant genome as part of the natural infection the plant genome as part of the natural infection process. process.

Any foreign DNA inserted into the T-DNA will also be Any foreign DNA inserted into the T-DNA will also be

integratedintegrated. .

Ti-plasmid based vectorsTi-plasmid based vectors

Binary systems Co-integrated vectors

Needs 2 vectors: Needs 3 vectors

Disarmed Ti plasmid with gene of interest(no vir genes)

Helper vectorfor infection(with vir genes)

Disarmed Ti plasmid capable for infection

Intermediate vector with T-region and gene of interest (transferred by conjugation)

Form co-integrated plasmidafter homologous recombination on T-DNA

Helper vectorfor transfer of intermediate plasmid into A.tum

Co-integrated vectors Co-integrated vectors (hybrid ti-plasmids)(hybrid ti-plasmids)

DISADVANTAGES: 1) Long homologies required between the Ti plasmid and the E. coli plasmids (pBR322 based Intermediate vectors) making them difficult to engineer and use

2) Relatively inefficient gene transfer compared to the binary vector

In a modern labsrarely used

Ti plasmid vector systems Ti plasmid vector systems are often working as binary vectorsare often working as binary vectors

DISADVANTAGE: Depending on the orientation, plasmids with two different origins of replication may be unstable in E. coli.

ADVANTAGE: small vectors are used, which increases transfer efficiency from E. coli to Agrobacterium. No intermolecular recombination is needed

Promoters useful for expression Promoters useful for expression in transgenic plantsin transgenic plants

35S, cauliflower mosaic virus 35S promoter

CaMoV 35S is a strong promoter that is active

in essentially all dicot plant tissues.

CaMoV is a circular dsDNA genome virus

Phenolics Produced by Wounded Plant cell

PLANT CELL

AGROBACTERIUM

VirA VirG

Phenolics detected by the VirA/VirG two component sensor system.

Vir Gene expression induced

T-DNA

Vir D1 & D2 cut T-DNA at right and left borders.

VirD2 attaches to exposed 5I end

VIP1 associates with the complex to target it to the nucleusVIP2 associates the complex to transcriptionally active DNAT-DNA integrates into plant DNA

and gall production is initiated.

Gall Formation!

Formation of T-complex

Bacterial Plasmid

Formation of T-Pilus

Procedure for creation a transgenic plantProcedure for creation a transgenic plant

1. Both plasmids are transfected into A.tumefaciens

2. Plant cell culture is infected with A.tumefaciens

3. Products of Vir genes excised gene of interest within T-DNAand transfer it to plant chromosome

Polylinker Kan-resistance geneT-DNA Repeat T-DNA Repeat

Gene of interest

4. Plant cells are selected on kanamycine

5. Presence of transgene confirmed by PCR

6. Whole plant could be grown from transformed cells !!!

Transgenic PlantsTransgenic Plants Currently 16 countries permit the cultivation of transgenic cropsCurrently 16 countries permit the cultivation of transgenic crops 99% of the total acreage is in 6 countries99% of the total acreage is in 6 countries

USA, China, Argentina, Canada, Brazil* & Australia USA, China, Argentina, Canada, Brazil* & Australia Five crops are currently in release Five crops are currently in release

soybean, cotton, corn, canola, squash soybean, cotton, corn, canola, squash

tomato, potato and flax in pasttomato, potato and flax in past

Since the first widespread release in 1996, land devoted to transgenic crops Since the first widespread release in 1996, land devoted to transgenic crops has increased by 10 - 50% yearly has increased by 10 - 50% yearly

90% of Canadian canola is GMO - mostly transgenic90% of Canadian canola is GMO - mostly transgenic 20+ countries suspected of growing transgenic crops without official 20+ countries suspected of growing transgenic crops without official

approvalapproval

Global area of transgenic cropsGlobal area of transgenic crops(ISAA Brief. Global Review of Commercialised Transgenic crops: 1998 & 2001)(ISAA Brief. Global Review of Commercialised Transgenic crops: 1998 & 2001)

Acreage of transgenic Acreage of transgenic crops has gone from crops has gone from nothing in 1995 to nothing in 1995 to around 135 million around 135 million

acres in 2001acres in 2001..

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Approved Transgenic plantsApproved Transgenic plants

SoybeanSoybean Corn Corn CottonCotton Oil Seed rapeOil Seed rape SugarbeetSugarbeet SquashSquash TomatoTomato TobaccoTobacco

CarnationsCarnations PotatoPotato FlaxFlax PapayaPapaya ChicoryChicory RiceRice MelonMelon

Types of GM crops (1998)Types of GM crops (1998)

Soybean and corn are the Soybean and corn are the major GM cropsmajor GM crops

* Large acreageLarge acreage* Grown in the USAGrown in the USA* Can be regeneratedCan be regenerated Acreage of potatoes is small Acreage of potatoes is small

(<0.1 million hectares)(<0.1 million hectares)

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Types of genetic modificationTypes of genetic modification

>99% of all transgenic >99% of all transgenic crops are either crops are either herbicide or insect herbicide or insect resistantresistant

<1% have other traits<1% have other traits

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